The present invention relates to antibodies against human CCN1 (CCN1 antibody), methods for their production, pharmaceutical compositions containing said antibodies, and uses thereof.
CCN1 (CYR61, GIG-1, IGFBP-10, SwissProt 000622) is a growth factor-inducible immediate-early gene, a member of the CCN protein family and is involved in cell adhesion, angiogenesis, apoptosis and either growth arrest or growth stimulation (reviewed in Chen Y. and Xiao-Yan D., J. Cell. Biochem. 100 (2007) 1337-1345 and Kubota, K. and Tagikawa, M., Angiogenesis 10 (2007) 1-11). CCN1 consists of a modular structure and contains 38 conserved cysteine residues. CCN1 shares a common modular structure with the other CCN proteins (except for CCN5 lacking the C-terminal module). CCN1 comprises an insulin-like growth factor-binding protein (IGFBP)-like motif (domain I, IGF-BP, amino acids 26-97), a von Willebrand type C domain (domain II, VWC, amino acids 98-164), a thrombospondin type I domain (domain III, TSP, amino acids 228-273) and a C-terminal module (domain IV, CT, amino acids 286-360). Between domain II and III a variable domain (Var, amino acids 165-227) is located. CCN1 binds to, and functions through, integrin αvβ3 to promote pro-angiogenic activity (Chen, N. et al., J. Biol. Chem. 42 (2004) 44166-44176). Integrin αvβ3 binding sites of CCN1 are between amino acids 116-135 (Chen N. et al., J. Biol. Chem. 42 (2004) 44166-44176) and in domain III (Leu, S. J. et al., J. Biol. Chem. 278 (2003) 33801-33808). Integrin α6β1 and heparin binding sites are located in domain III and IV mediating cellular (Chen et al., J. Biol. Chem. 276 (2001) 47329-47337).
Although CCN proteins share an insulin-like growth factor-binding protein (IGFBP)-like motif close to the N terminus, no clear experimental significance exists to suggest a function in the IGF signaling pathway (Grotendorst, G. R. et al., Endocrinology 141 (2000) 2254-2256). The von Willebrand type C domain (VWC), the thrombospondin type I domain, and the C-terminal module (which is absent in WISP2/CCN5) are considered to be important for protein-protein interactions, either oligomerization (VWC) or interactions with extracellular matrix molecules and receptors.
Mouse CCN1 shares 91% amino acid sequence identity with human CCN1. Murine CCN1 and antibodies against murine CCN1 are known from O'Brien, T. P. et al., Mol. Cell. Biol. 10 (1990) 3569-3577). There is also a high homology between CCN1 and connective tissue growth factor-II (CTGF 2/CTGH 2). Further (but lower) homologies were found with NOV and FISB-12. Babic, A. M. et al., in Proc. Natl. Acad. Sci. USA 95 (1998) 6355-6360, describe that their antibodies against human CCN1 show a cross-reactivity with mouse CCN1 but not with FISB-12.
CCN1 and antibodies against CCN1 are mentioned in WO 96/001896, relating to Connective tissue growth factor-II” (CTGF-2), WO 97/033995, relating to human CCN1, WO 01/55210, relating to CCN1 compositions and methods, WO 2005/040191, relating to CCN1 Compositions and Methods, WO 02/04480, relating to Connective tissue growth factor-II” (CTGH-2), WO 01/98359, relating to CCN1 as a target for treatment and diagnosis of breast cancer, WO 02/26193, relating to the use of CCN1 in the treatment and diagnosis of human uterine leiomyomas. Grotendorst, G. R. and Duncan, M. R., FASEB J. 19 (2005) 729-738 describe antibodies against domains of CTGF and found some activity for antibodies against the N-terminal and C-terminal domains. As the domains were generated by plasmin cleavage, this cleavage occurs within the variable domain and said domain was destroyed.
Dong Xie et al., in J. Biol. Chem. 276 (2001) 14187-14194, describe that CCN1 is associated with more advanced disease, and in Cancer Res. 64 (2004) 1987-1996, that CCN1 is overexpressed in gliomas and involved in integrin-linked kinase-mediated akt and β-catenin-TCF/Lef signaling pathways.
Schuetze, N. et al., Protein Expr. Purif. 42 (2005) 219-225 relates to the expression, purification, and functional testing of recombinant CCN1. Schuetze cloned the open reading frame into a baculovirus expression vector and transfected the construct into SF-21 insect cells. Recombinant CCN1 was expressed as a fusion protein with the Fc-domain of human IgG and purified using affinity chromatography on protein G-Sepharose columns. As CCN1 possesses 10% cysteine residues and therefore represents a very adhesive protein which could be easily lost during purification and handling. Also the protein tends to form aggregates and to precipitate during or after purification. The addition of the Fc-tag minimized these difficulties. The authors were not sure whether the Fc-tag of the rCCN1 protein alters or influences the biochemical characteristics of the protein. Leu, S. J. et al., J. Biol. Chem. 278 (2003) 33801-33808 describe the recombinant expression of hexahistidine tagged CCN1 fragments of domain I (IGFBP), domain II (VWC) and domain III (TSP1). A native untagged protein is not available.
Jedsadayanmata, A. et al., J. Biol. Chem. 274 (1999) 24321-24327 mention anti-peptide polyclonal antibodies raised against a peptide comprising parts of the variable domain (a.a. 163 to 229) and TSP1 domain (a.a. 228-273) of CCN1 for investigation of activation-dependent adhesion of human platelets. The polyclonal antiserum was generated by immunizing rabbits with a Var-GST fusion protein which was recombinant produced in E. coli (Kireeva et al., Exp. Cell Res. 233 (1997) 63-77). U.S. Pat. No. 7,521,540 claims an antibody binding to amino acids 163-229 and 210-225 of human Cyr61. According to U.S. Pat. No. 7,521,540 a polyclonal Cyr61-specific antiserum, using a construct fusing amino acids 163 to 229 of Cyr61 to GST was made by immunization of New Zealand white rabbits.